Methods and compositions for enhancing cartilage repair

ABSTRACT

The invention relates to methods of enhancing repair of a cartilage and/or inducing formation of a cartilage by administering a cell, which expresses a factor of the T-box family, which includes inter-alia the brachyury. In another embodiment, the invention relates to an engineered cell, which is transfected with a vector comprising a nucleic acid sequence encoding a factor of the T-box family, thereby expressing a factor of the T-box family. In another embodiment, the invention relates to compositions comprising a vector, which comprises a nucleic acid sequence encoding a factor of the T-box family and in another embodiment the composition-comprising cell that expresses a factor of the T-box family, which includes inter-alia the brachyury.

This application is a Continuation-in Part Application of U.S. Ser. No.10/067,980, filed Feb. 8, 2002, which claims the benefit of U.S. Ser.No. 09/376, 276, filed Aug. 2, 1999, now abandoned, which claimspriority from DE Application No. 198.37.438.0, filed Aug. 18, 1998,which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention provides methods of enhancing repair of a cartilage and/orinducing formation of a cartilage by contacting a cell, which expressesat least one factor of the T-box family. In another embodiment, theinvention provides an engineered cell, which is transfected with avector comprising at least one nucleic acid sequence encoding a factorof the T-box family, thereby expressing at least one factor of the T-boxfamily. This invention provides a composition comprising a vector, whichcomprises at least one nucleic acid sequence encoding a factor of theT-box family.

BACKGROUND OF THE INVENTION

The meniscus, fibrocartilaginous tissue found within the knee joint, isresponsible for shock absorption, load transmission, and stabilitywithin the knee joint. According to the National Center for HealthStatistics, over 600,000 surgeries each year are the result ofcomplications with the meniscus. The meniscus has the intrinsic abilityto heal itself; unfortunately, this property is limited only to thevascular portions of the tissue. For damage outside of these areas andoverall degeneration of the tissue, methods need to be developed thatwill assist the meniscus in healing itself Sweigart M A Tissue Eng 7(2),111-29 (April 2001);

Degeneration of articular cartilage in osteoarthritis is a seriousmedical problem caused by arthritis, both rheumatoid and osteoarthritis.Drugs are given to control the pain and to keep the swelling down, butthe cartilage continues to be destroyed. Eventually, the joint must bereplaced. It is still unknown why cartilage does not heal and nosolutions to this problem are known Mankin, N. E. J. Med. 331(14),940-941 (October 1994). Soon after superficial injury, chondrocytesadjacent to the injured surfaces show a brief burst of mitotic activityassociated with an increase in glycosaminoglycan and collagen synthesis.Despite these attempts at repair, there is no appreciable increase inthe bulk of cartilage matrix and the self-repair process is usuallyineffective in healing the defects.

Osteochondral, or full-thickness, cartilage defects expand into thesubchondral bone. Such defects arise after the detachment ofosteochondritic dissecting flaps, fractured osteochondral fragments, orfrom chronic wear of degenerative articular cartilage. Osteochondraldefects depend on the extrinsic mechanism for repair. Extrinsic healingrelies on mesenchymal elements from subchondral bone to participate inthe formation of new connective tissue. This fibrous tissue may or maynot undergo metaplastic changes to form fibrocartilage. Even iffibrocartilage is formed, it does not display the same biochemicalcomposition or mechanical properties of normal articular cartilage orsubchondral bone and degenerates with use, Furukawa, et al., J. BoneJoint Surg. 62A, 79 (1980); Coletti, et al., J. Bone Joint Surg. 54A,147 (1972); Buckwalter, et al., “Articular cartilage: composition,structure, response to injury and methods of facilitating repair”, inArticular Cartilage and Knee Joint Function: Basic Science andArthroscopy, Ewing J E, Ed., (New York, Raven Press, 1990), 19.

Injection of dissociated chondrocytes directly into the site of thedefect has also been described as a means for forming new cartilage, asreported by Brittberg, et al., N. E. J. Med. 31, 889-895 (October 1994).Cartilage was harvested from minor load-bearing regions on the uppermedial femoral condyle of the damaged knee, cultured, and implanted twoto three weeks after harvesting.

Moreover, if the defect includes a part of the underlying bone, this isnot corrected by the use of chondrocytes. The bone is required tosupport the new cartilage.

Cartilage grafts are also needed in plastic surgery like in rhinoplasty,and the reconstruction of ears.

The possibility of using stem cells was also examined. Stem cells arecells which are not terminally differentiated, which can divide withoutlimit, and divide to yield cells that are either stem cells or whichirreversibly differentiate to yield a new type of cell. Unfortunately,there is no known specific inducer of the mesenchymal stem cells thatyields only cartilage.

In vitro studies in which differentiation is achieved using differentbioactive factors or molecules, yields differentiation of the cells tocartilage which eventually calcified and turned into bone.

Thus, there is a need to have a method and composition for the formationor repair of a cartilage or a bone. In another embodiment, it will behighly advantageous to have a cell, which can divide and form acartilage or a bone tissue.

SUMMARY OF THE INVENTION

In one embodiment the invention provides a method of enhancing repair ofa cartilage comprising the step of administering to a subject aneffective amount of a cell which expresses at least one factor of theT-box family, thereby enhancing repair of the cartilage.

In another embodiment the invention provides a method of inducingformation of a cartilage comprising the step of administering to asubject an effective amount of a cell which expresses at least onefactor of the T-box family, thereby inducing formation of the cartilage.

In another embodiment the invention provides a method of enhancingrepair of a cartilage in the body comprising the step of administratinga recombinant vector which comprises a nucleic acid encoding a factor ofthe T-box family to the cartilage of a subject, thereby enhancing repairof the cartilage.

In another embodiment the invention provides a method of inducingformation of a cartilage in the body comprising the step ofadministrating a recombinant vector which comprises a nucleic acidencoding a factor of the T-box family to the cartilage of a subject,thereby inducing formation of the cartilage.

In another embodiment the invention provides a method of inducingchondrocyte differentiation comprising the step of administering of arecombinant vector, which comprises a nucleic acid encoding a factor ofthe T-box family, thereby inducing chondrocyte formation.

In another embodiment the invention provides a method of repairing orforming a cartilage in a subject in need comprising the steps of:obtaining a cell from of the subject; transfecting said cell with arecombinant vector comprising a nucleic acid sequence encoding a factorof the T-box family, so as to obtain an engineered cell which expressesa factor of the T-box family; and administering said engineered cell tothe subject.

In another embodiment the invention provides a method for the productionof transplantable cartilage matrix, the method comprising the steps of:obtaining a cell; transfecting said cell with a recombinant vectorcomprising a nucleic acid sequence encoding a factor of the T-boxfamily, so as to obtain an engineered cell which expresses a factor ofthe T-box family; and culturing said cell with the cell-associatedmatrix for a time effective for allowing formation of a transplantablecartilage matrix.

In another embodiment the invention provides an engineered cell, whichexpresses a factor of the T-box family.

In another embodiment the invention provides an implant devicecomprising at least one engineered cell which expresses a factor of theT-box family and a pharmaceutically acceptable carrier.

In another embodiment the invention provides a composition comprising anengineered cell which expresses a factor of the T-box family and apharmaceutically acceptable carrier.

In another embodiment the invention provides a composition comprising atleast one recombinant vector which comprises a nucleic acid sequenceencoding at least one factor of the T-box family and a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FGFR3 mediates chondrocytic differentiation in mesenchymal stemcell line C3H10T1/2. FIG. 1 a shows the RT-PCR analyses ofBMP2-dependent expression of FGF- and PTH/PTHrP-receptors in mesenchymalstem cell line C3H10T1/2 in the presence or absence of recombinantllyexpressed BMP2. FIG. 1 b shows the effect of cyclohexamide pretreatmentof C3H10T1/2 cells. Cycloheximide pre-treatment of C3H10T/1/2 cells doesnot prevent BMP-induction of the FGFR3 gene. Cells were mock-treated(control) or were treated with BMP2 (50 ng/ml). FIG. 1 c demonstrateswestern immunoblotting for the detection of BMP2-dependent FGFR3 andFGFR2 expression in cellular extracts of C3H10T1/2 lines. FIG. 1 d leftpanel: western immunoblotting to demonstrate the forced expression ofFGFR3 in mesenchymal progenitors C3H10T1/2; right panel: the recombinantexpression of FGFR3 in C3H10T1/2 leads to enhanced levels of activatedMAP-kinases pERK-1 and pERK-2 during cultivation. Cell lysates wereprepared 0 (=confluence) and 4 days post-confluence. FIG. 1 edemonstrates that the forced expression of FGFR3 in parental C3H10T1/2cells is sufficient for the induction of the chondrogenic lineage.

FIG. 2. The T-box transcription factor Brachyury mediates chondrogenicdifferentiation in MSCs in vitro and ectopically in vivo. FIG. 2 a upperpanel: schematic representation of Brachyury according to Kispert etal., 1995 (1995). FIG. 2 a lower panel: western immunoblotting ofrecombinant HA-tagged Brachyury (aa 1-436) in cellular extracts ofC3H10T1/2 (C3H10T1/2-Brachyury) with HA-antibody SC-805 (Santa Cruz)Brachyury has been constitutively expressed under the control of themurine PGK-promoter. Expression of Brachyury is indicated (triangle).Molecular weight marker (M) shown is ovalbumin (43 kDa). FIG. 2 b showsthe histological characterization of C3H10T1/2-Brachyury cells inculture. Upper panel: at day 4 post-confluency cells developalkaline-phosphates positive osteoblast-like cells. Lower right panel:Alcian Blue histology of C3H10T1/2 cells stably expressing Brachyuryindicative for secreted proteoglycans and efficient differentiation intothe chondrogenic lineage. Lower left panel:Collagen-immunohistochemistry of C3H10T1/2-Brachyury cells in culture 7days post-confluency. FIG. 2 c shows the RT-PCR analysis of theexpression of chondrogenic and osteogenic marker genes in C3H10T1/2cells recombinantlly-expressing Brachyury. FIG. 2 d shows the forcedexpression of the T-box factor Brachyury in C3H10T1/2 cells which leadsto differentiation into chondrocytes and cartilage development at murineectopic sites after intramuscular transplantation.

FIG. 3. Dominant-negative Brachyury (dnBrachyury; T-box domain) blocksBMP2-mediated chondrogenic development in C3H10T1/2 MSCs in vitro andectopically in vivo. FIG. 3 a shows that Brachyury's T-box domaininterferes with the transcriptional activity of full-length Brachyury.FIG. 3 b shows expression of dnBrachyury (T-box domain) inC3H10T1/2-BMP2 during cultivation (day 0; cellular confluence) The T-boxdomain (aa 1-229) has been subcloned and HA-tagged in expression vectorpMT7T3 and constitutively expressed in C3H10T1/2-BMP2 cells. Therecombinantlly expressed T-box domain (dnBrachyury) is indicated(triangle). FIG. 3 c demonstrates RT-PCR experiments withosteo-/chondrogenic marker genes show that T-box domain (dnBrachyury)expression in C3H10T1/2-BMP2 cells interferes with the BMP2 dependent ofFGFR2 but not FGFR3 expression. FIG. 3 d shows that dnBrachyury (T-box)interferes with BMP2-mediated FGFR2 expression as analyzed by westernimmunoblotting with antiFGFR3 and antiFGFR2 antibodies as describedFIG. 1. FIG. 3 e shows the forced expression of the dominant-negativeacting T-box domain in C3H10T1/2-BMP2 cells interferes with BMP-2mediated osteo-/chondrogenic development.

FIG. 4. Dominant-negative FGFR3 (dnFGFR3) interferes withosteo-/chondrogenic development, with FGFR2- and withBrachyury-expression in C3H10T1/2-BMP2. FIG. 4 a shows that forcedexpression of dnFGFR3 in C3H10T1/2-BMP2 cells interferes with BMP-2mediated development of alkaline phosphatase positive and Alcian Bluepositive chondrocyte-like cells, respectively. FIG. 4 b shows thatdnFGFR3 interferes with BMP2-dependent FGFR2 and Brachyury but not withFGFR3 expression in C3H10T1/2-BMP2 cells.

FIG. 5. FGFR3 and Brachyury are involved in an auto regulatory loop.FIG. 5 a shows RT-PCR analyses of FGFR3 and Brachyury mRNA levels inmesenchymal progenitors C3H10T1/2 expressing recombinant FGFR3(C3H10T1/2-FGFR3) or Brachyury (C3H10T1/2-Brachyury). FIG. 5 b showsthat Smad1-signaling is not sufficient for Brachyury and FGFR3 but forosteocalcin expression. RT-PCR analyses of FGFR3 and Brachyury mRNAlevels in mesenchymal progenitors C3H10T1/2 expressing the biologicallyactive Smad1-MH2 domain (C3H10T1/2-Smad1-MH2).

FIG. 6. Brachyury is expressed at skeletal sites during late murineembryonic development (18.5 dpc). Comparative expression analysis ofmurine Brachyury (Bra), Collagen 1a1 (Col 1a1) and Collagen 2a1 (Col2a1) in embryonic development 18.5 dpc. a, Intervertebral discsdevelopment. Consecutive sagittal (a-g) and transversal (h-j) sectionsof 18.5 dpc mouse embryos were hybridized with riboprobes as indicated.Expression of Brachyury is enhanced in the nucleus pulposus (a, d), Col1a1 in the outer annulus (arrowheads in b, e), and Col 2a1 in thecartilage primordium of the vertebrae (c,f). No signals are obtainedusing RNase pre-incubated sections (g). With transversal sections at thelevel of the upper lumbar vertebra expression of Brachyury is inaddition detectable in distinct cells of the neural arch (h) whereas Col1a1 is expressed in the outer annulus (i) and Col 2a1 in the cartilageprimordium (j). b, Limb bud development. Consecutive transversalsections of a 18.5 dpc mouse hind limb at the level of the metatarsalshybridized with riboprobes as indicated. Expression of Brachyury isevident in distinct chondrogenic cells of the forming metatarsal bones(a), better visible with higher magnification (b,c). In contrast Col 1a1is expressed in the outer periosteal layer (d-f) and Col 2a1 expressionis enhanced in differentiating chondrocytes (g-i). As it was evident forthe intervertebral disc formation expression of Brachyury is onlyevident in chondrocyte-like cells that do not express-Col 2a1. ch,chondrocytes; cp, cartilage primordium; mta, metatarsal; np, nucleuspulposus; oa, outer annulus; pl, periosteal layer; sk, skin bar, 100 □m

FIG. 7. Model of BMP2-dependent osteo-/chondrogenic development inmesenchymal stem cells.

FIG. 8. Human adult MSCs (3×106) were transfected with 30 ug ofBrachyury plasmid. 24 hours post transfection, RNA was isolated andRT-PCR was performed using specific primers to the Brachyury cDNA. 20 ulof the PCR reaction sample were loaded and electrophoresed in 2% Agarosegel. The gel image demonstrated positive Brachyury expression (Positivecontrol-Brachyury plasmid).

DETAILED DESCRIPTION OF THE EMBODIMENTS

In one embodiment, the invention relates to methods of enhancing repairof a cartilage and/or inducing formation of a cartilage by administeringa cell, which expresses a factor of the T-box family, which includesinter-alia the brachyury. In another embodiment, the invention relatesto an engineered cell, which is transfected with a vector comprising anucleic acid sequence encoding a factor of the T-box family, therebyexpressing a factor of the T-box family. In another embodiment, theinvention relates to compositions comprising a vector, which comprises anucleic acid sequence encoding a factor of the T-box family and inanother embodiment the composition-comprising cell that expresses afactor of the T-box family, which includes the brachyury.

The term “cartilage” refers hereinabove to a specialized type of denseconnective tissue consisting of cells embedded in a matrix. There areseveral kinds of cartilage. Translucent cartilage having a homogeneousmatrix containing collagenous fibers is found in articular cartilage, incostal cartilages, in the septum of the nose, in larynx and trachea.Articular cartilage is hyaline cartilage covering the articular surfacesof bones. Costal cartilage connects the true ribs and the sternum.Fibrous cartilage contains collagen fibers. Yellow cartilage is anetwork of elastic fibers holding cartilage cells which is primarilyfound in the epiglottis, the external ear, and the auditory tube.Cartilage is tissue made up of extracellular matrix primarily comprisedof the organic compounds collagen, hyaluronic acid (a proteoglycan), andchondrocyte cells, which are responsible for cartilage production.Collagen, hyaluronic acid and water entrapped within these organicmatrix elements yield the unique elastic properties and strength ofcartilage.

As used herein, “hyaline cartilage” refers to the connective tissuecovering the joint surface. By way of example only, hyaline cartilageincludes, but is not limited to, articular cartilage, costal cartilage,and nose cartilage.

As used herein, the term “enhancing cartilage repair” refers to healingand for regeneration of cartilage injuries, tears, deformities ordefects, and prophylactic use in preventing damage to cartilaginoustissue.

As used herein, the term “inducing formation” refers to the use incartilage renewal or regeneration so as to ameliorate conditions ofcartilage, degeneration, depletion or damage such as might be caused byaging, genetic or infectious disease, accident or any other cause, inhumans, livestock, domestic animals or any other animal species. Inanother embodiment the formation of a cartilage is required forcartilage development in livestock, domestic animals or any other animalspecies in order to achieve increased growth for commercial or any otherpurpose. In another embodiment the formation of a cartilage is requiredin plastic surgeries, such as without being limited facialreconstruction in order to obtain a stabilized shape.

In one embodiment there is provided a recombinant vector comprising anucleic acid sequence encoding a factor of the T-box family.

The term “T-box family” defined as a family of transcription factorsthat share the T-box, a 200 amino acid DNA-binding domain (T-box, aa1-229). The T-box family has been identified in both vertebrates and invertebrates and plays a key role in embryonic development. The T- boxfamily further includes variant and fragments of the T-box familytranscription factors.

The T-Box gene family can be said to consist of several genericentities: T, Tbr-1, Tbx1-9, 11, 12, 17 and T2 and many species has beenshown to contain orthologs. Several mouse T-Box genes have beenreported; mu-T, mu-Tbr1 (identified in a subtractive hybridizationscreen for genes specifically involved in regulating forebraindevelopment (Bulfone et al. (1995) Neuron 15:63-78), mu-Tbx1-6, mm-Tbx13(Wattler et at., Genomics 48:24-33), and mm-Tbx14 (Wattler et al. (1998)Genomics 48:24-33, 1998). There are four Xenopus genes (Xbra, x-eomes,x-ET and x-VegT (Zhang et al. (1996) Development 122:4119-4129; Smith etal. (1995) Semin Dev Biol 6:405-410; Lustig et al. (1996) Development122:4001-4012; Stennard et al. (1996) Development 122:4179-4188; Horb etal. (1997) Development 124:1689-1698; Ryan et al. (1996) Cell87:989-1000). Human orthologs for six of eight mouse genes have beenidentified. Hu-T (Edwards et al. (1996) Genome Res 6:226-233; Morrisonet al. (1996) Hum Mol Genet 5:669-674) and hu-TBRI (Bulfone et al.(1995) Neuron 15:63-78) were found by homology with the mouse orthologs.Hu-TBX2 was isolated independently by two groups from embryonic kidneycDNA libraries (Campbell et al. (1995) Genomics 28:255-260; Law et al.(1995) Mamm Genome 6:267-277). Hu-TBX1, hu-TBX3, and hu-TBX5 were foundduring investigations aimed at uncovering the genetic basis of humandevelopmental dysmorphic syndromes and were recognized as orthologs ofthe mouse genes by sequence homology (Li et al. (1997) Nat Genet15:21-29; Basson et al. (1997) Nat Genet 15:30-35; Chieffo el al.(1997)Genome 43:267-277).

There is currently only a handful of known mutations in T-Box genes.Spontaneous mutations in hu-TBX3 (Bamshad et al. (1997) Nat Genet16:311-315) and hu-TBX5 (Li et al. (1997) Nat Genet 15:21-29; Basson etal. (1997) Nat Genet 15:30-35) have been reported. These mutations atT-Box genes play a role in several human autosomal, dominantdevelopmental syndromes: Ulnar-Mammary syndrome and Holt-Oram syndrome.Ulnar-Mammary syndrome is characterized by limb defects, abnormalitiesof apocrine glands such as the absence of breasts, axillary hair andperspiration, dental abnormalities such as ectopic, hypoplastic andabsent canine teeth, and genital abnormalities such as micropenis, shawlscrotum and imperforate hymen. Holt-Oram syndrome is characterized bycardiac septal defects and preaxial radial ray abnormalities of theforelimbs (Li el al. (1997) Nat Genet 15:21-29; Basson et al. (1997) NatGenet 15:30-35; Bamshad et al. (1997) Nat Genet 16:311-315). Mutationsin the 5′ end of TBX5 lead to substantial cardiovascular malformationsand relatively mild skeletal defects while mutations in the 3′ end ofthe gene cause severe skeletal malformation and have less effect oncardiac development (McCarthy, M (1998) Lancet 351(9115):1564; Basson,C. T. et al (1997) Nature Genetics 15:30-35). A better understanding ofthe role which T-Box transcription factors play in embryogenesis,organogenesis and organ regeneration has been recently recognized. T-Boxrelated genes have been found in many species, making up a large groupof T-Box transcription factors which are highly conserved in theirDNA-binding capacity but may be highly divergent in the non-DNA-bindingregions. There are common features which define the family, as well asspecific differences that define individual members. Phylogeneticanalysis suggests that the genome of most animal species will have atleast five T-Box genes (related to mu-Tbx2, mu-Tbx, mu-Tbx1, mu-T, andmu-Tbr1). There are at least 16 distinct members in 11 different animalgroups that have been reported and human orthologs of six of the eightmouse genes have already been identified.

In another embodiment, the vector comprising a nucleic acid whichencodes for Brachyury, or T, which refers hereinabove to the founderfactor of the T-box family.

The immediate BMP2-dependent upregulation of FGFR3 in MSCs (C3H10T1/2)and the inherent capacity of this receptor to initiate chondrogenicdevelopment in these cells prompted a screen for FGFR3-regulatedtranscription factors. The chondrogenic potential of Brachyury afterrecombinant expression in wild-type C3H10T1/2 cells has been shown, bythe use of a subtractive screening method, exemplified in Example 1that, among the transcription factors tested, the T-box transcriptionfactor Brachyury was upregulated in FGFR3-expressing C3H10T1/2 cells(see also FIG. 5 a).

As used herein, the term “nucleic acid” refers to polynucleotides or toologonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA) or mimetics thereof. The term shouldalso be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs, and, as applicable to the embodimentbeing described, single (sense or antisense) and double-strandedpolynucleotides. This term includes oligonucleotides composed ofnaturally occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

The vector molecule can be any molecule capable of being delivered andmaintained within the target cell or tissue such that the gene encodingthe product of interest can be stably expressed. The vector moleculepreferably utilized in the present invention is either a viral orretroviral vector molecule or a plasmid DNA non-viral molecule. Thismethod preferably includes introducing the gene encoding the productinto the cell of the mammalian connective tissue for a therapeutic orprophylactic use. Unlike previous pharmacological efforts, the methodsof the present invention employ gene therapy to address the chronicdebilitating effects of joint pathologies. The viral vectors used in themethods of the present invention can be selected form the groupconsisting of (a) a retroviral vector, such as MFG or pLJ; (b) anadeno-associated virus; (c) an adenovirus; and (d) a herpes virus,including but not limited to herepes simplex 1 or herpes simples 2 or(e) lentivirus. Alternatively, a non-viral vector, such as a DNA plasmidvector, can be used. Any DNA plasmid vector known to one of ordinaryskill in the art capable of stable maintenance within the targeted cellor tissue upon delivery, regardless of the method of delivery utilizedis within the scope of the present invention. Non-viral means forintroducing the gene encoding for the product into the target cell arealso within the scope of the present invention. Such non-viral means canbe selected from the group consisting of (a) at least one liposome, (b)Ca3 (PO4) 2, (c) electroporation, (d) DEAE-dextran, and (e) injection ofnaked DNA.

As will be appreciated by one skilled in the art, a fragment orderivative of a nucleic acid sequence or gene that encodes for a proteinor peptide can still function in the same manner as the entire, wildtype gene or sequence. Likewise, forms of nucleic acid sequences canhave variations as compared with the wild type sequence, while thesequence still encodes a protein or peptide, or fragments thereof, thatretain their wild type function despite these variations. Proteins,protein fragments, peptides, or derivatives also can experiencedeviations from the wild type from which still functioning in the samemanner as the wild type form. Similarly, derivatives of the genes andproducts of interest used in the present invention will have the samebiological effect on the host as the non-derivatized forms. Examples ofsuch derivatives include but are not limited to dimerized oroligomerized forms of the genes or proteins, as wells as the genes orproteins modified by the addition of an immunoglobulin (Ig) group.Biologically active derivatives and fragments of the genes, DNAsequences, peptides and proteins of the present invention are thereforealso within the scope of this invention. In addition, any nucleic acid,which is cis acting and integrated upstream to an endogenous factor ofthe T-box family nucleic acid sequence, is relevant to the presentinvention.

The term “cis-acting” is used to describe a genetic region that servesas an attachment site for DNA-binding proteins (e.g. enhancers,operators and promoters) thereby affecting the activity of genes on thesame chromosome.

It was shown that Brachyury expression is upregulated by certain factorsuch as BMP2 and/or FGF3 (see FIGS. 4 and 5). Thus, in anotherembodiment, the vector of the invention further comprises a nucleicacid, which encodes to a protein, which activated the BMP signalingpathway. In another embodiment, the protein, which activated the BMPsignaling pathway, is a member of the BMP family. In another embodimentthe BMP is a BMP2. In another embodiment the vector further comprising anucleic acid for fibroblast growth factor namely FGF-3.

The term “protein which activates BMP mediated signaling pathway” isdefined hereinabove as a protein that can activate the BMP receptors, orthe signaling cascade down stream of the receptor to elicit BMP specificcellular response. Examples, without being limited are members of theBMP family, such as the BMP proteins BMP-1, BMP-2, BMP-3, BMP-4, BMP-5,BMP-6 and BMP-7, disclosed for instance in U.S. Pat. Nos. 5,108,922;5,013,649; 5,116,738; 5,106,748; 5,187,076, and 5,141,905; BMP-8,disclosed in PCT publication W091/18098; BMP-9, disclosed in PCTpublication W093/00432; and BMP-10 or BMP-11, disclosed in co-pendingpatent applications, Ser. No. 08/061,695 presently abandoned, acontinuation-in-part of which has issued as U.S. Pat. No. 5,637,480, and08/061,464 presently abandoned, a continuation-in-part of which hasissued as U.S. Pat. No. 5,639,638 filed on May 12, 1993.

In another embodiment the vector may include also nucleic acids encodingother therapeutically useful agents including MP52, epidermal growthfactor (EGF), fibroblast growth factor (FGF), platelet derived growthfactor (PDGF), transforming growth factors (TGF-α and TGF-β), andfibroblast growth factor-4 (FGF-4), parathyroid hormone (PTH), leukemiainhibitory factor (LIF/HILDA/DIA), insulin-like growth factors (IGF-Iand IGF-II). In another embodiment, the vector comprises nucleic acidencoding an anti-inflammatory agent such as IL1 receptor antagonist, orIL4 or IL10 agonist.

In another embodiment the cell of the invention further expressestherapeutically useful agents including MP52, epidermal growth factor(EGF), fibroblast growth factor (FGF), platelet derived growth factor(PDGF), transforming growth factors (TGF-α and TGF-β), and fibroblastgrowth factor-4 (FGF-4), parathyroid hormone (PTH), leukemia inhibitoryfactor (LIF/HILDA/DIA), insulin-like growth factors (IGF-I and IGF-II).In another embodiment, the vector comprises nucleic acid encoding ananti-inflammatory agent such as IL1 receptor antagonist, or IL4 or IL10agonist.

In another embodiment, there is provided an engineered cell, whichexpresses at least one factor of the T-box family.

The term “engineered cell” is defined hereinabove to a cell or to atissue, which had been genetically modified and is expressing a factorof the T-box family or in another embodiment, increased amounts of thefactor of the T-box family or in another embodiment express Brachury.The term “increased amount of the factor or the at least one factor ofthe T-box family refers hereinabove to at least 10 times more thannormal.

In one embodiment, the cell of the invention is a mammalian cell. Inanother embodiment, it is a mesenchymal stem cell, in another embodimentit is a progenitor cell, in another embodiment it is a cell derived froma cartilage. In another embodiment the cell can be derived from afibroblast cell line, a mesenchymal cell line, a chondrocyte cell line,an osteoblast cell line, or an osteocyte cell line. The fibroblast cellline may be a human foreskin fibroblast cell line or NIH 3T3 cell line.In another embodiment the cell of the invention is a synovial cell or asynoviocyte. Synoviocytes are found in joint spaces adjacent tocartilage have an important role in cartilage metabolism. Synoviocytesproduce metallo-proteinases, such as collagenases that are capable ofbreaking-down cartilage

Stem cells are defined as cells which are not terminally differentiated,which can divide without limit, and divides to yield cells that areeither stem cells or which irreversibly differentiate to yield a newtype of cell. Those stem cells which give rise to a single type of cellare call unipotent cells; those which give rise to many cell types arecalled pluripotent cells. Chondro/osteoprogenitor cells, which arebipotent with the ability to differentiate into cartilage or bone, wereisolated from bone marrow (for example, as described by Owen, J. CellSci. Suppl. 10, 63-76 (1988) and in U.S. Pat. No. 5,226,914 to Caplan,et al.).

It is important to note that mesenchymal stem cells and progenitors canbe isolated from different source tissues, skin, bone marrow, muscle,and liver. In addition any cell type with stem cell properties ordemonstrating differentiation plasticity for example without limitation,SP cells from the source of bone marrow, muscle, spleen or any othertissue.

Chondrogenic cells useful in the practice of the invention may beisolated from essentially any tissue containing chondrogenic cells. Asused herein, the term “chondrogenic cell” is understood to mean any cellwhich, when exposed to appropriate stimuli, may differentiate into acell capable of producing and secreting components characteristic ofcartilage tissue. The chondrogenic cells may be isolated directly frompre-existing cartilage tissue, for example, hyaline cartilage, elasticcartilage, or fibrocartilage. Specifically, chondrogenic cells may beisolated from articular cartilage (from either weight-bearing ornon-weight-bearing joints), costal cartilage, nasal cartilage, auricularcartilage, 30 tracheal cartilage, epiglottic cartilage, thyroidcartilage, arytenoid cartilage and cricoid cartilage. The cell from thecartilage can be derived from another animal, or another subject or inanother embodiment; the cell of the cartilage or the bone can be derivedfrom the subject in need.

In another embodiment, the cell further expresses at least one protein,which activates BMP mediated signaling pathway or a FGF protein.

The expression of the at least one factor of the T-box family incombination with either and BMP or FGF or both in the cell can be due tothe presence of two or more different vectors (trans vectors) or due tothe expression of one vector which comprises two or more differentnucleic acid sequences, which encode for the at least one member of theT-box family, the FGF and for at least one protein which activates theBMP mediated signaling pathway. It was shown that Brachyury'sDNA-binding domain without the associated regulatory domains (aa230-436) should dominant-negatively (dn) interfere with endogenousBrachyury-mediated events in C3H10T1/2-BMP2 cells see FIG. 3 b andexample 3.

In another embodiment the invention provides complex tissue engineering.This term refers to engineering a cell with different nucleic acidsequences, wherein each sequence encodes to a specific pathway ofdifferentiation. As such, the cell of the invention can be engineered todifferentiate to an osteoblast as well as to a chondrocyte.

In another embodiment there is provided a composition comprisingrecombinant vector comprising a nucleic acid sequence encoding the afactor of the T-box family and a pharmaceutically acceptable carrier. Itshould be noted that the term “a nucleic acid sequence encoding the afactor of the T-box family” refers hereinabove to “at least one nucleicacid sequence encoding the at least one factor of the T-box family”.Similarly the term “a cell” refers to “at least one cell”.

In another embodiment, there is provided a composition comprising atleast one engineered cell, wherein said engineered cell expresses leastone factor of the T-box family at least one protein and apharmaceutically acceptable carrier.

In another embodiment the composition can be a pharmaceuticalcomposition.

Compositions of the invention may further comprise additional proteins,such as additional factors. These compositions may be used to induce theformation or repair of cartilage tissue.

The compositions of the invention may comprise, also BMP-12 or VL-1(BMP-13), other therapeutically useful agents including MP52, epidermalgrowth factor (EGF), fibroblast growth factor (FGF), platelet derivedgrowth factor (PDGF), transforming growth factors (TGF-□ and TGF-□), andfibroblast growth factor-4 (FGF-4), parathyroid hormone (PTH), leukemiainhibitory factor (LIF/HILDA/DIA), insulin-like growth factors (IGF-Iand IGF-II). Portions of these agents may also be used in compositionsof the present invention. N another embodiment the composition comprisesanti-inflammatory agents such as IL1 receptor antagonists, or IL4 or IL10 agonists.

In another embodiment, there is provided an implant device fortransplantation in a subject in need comprising an engineered cell whichexpresses a factor of the T-box family and a pharmaceutically acceptablecarrier. Cartilage implants are often used in reconstructive or plasticsurgery such as rhinoplasty.

The preparation and formulation of such pharmaceutically/physiologicallyacceptable protein compositions, having due regard to pH, isotonicity,stability and the like, is within the skill of the art. The therapeuticcompositions are also presently valuable for veterinary applications dueto the lack of species specificity in factor of the T-box family due tohigh homology between species.

Particularly domestic animals and thoroughbred horses in addition tohumans are desired patients for such treatment with the compositions ofthe present invention.

The therapeutic method includes administering the composition topically,systemically, or locally as an injectable and/or implant or device. Whenadministered, the therapeutic composition for use in this invention is,of course, in a pyrogen-free, physiologically acceptable form. Further,the composition may desirably be encapsulated or injected in a viscousform for delivery to the site of tissue damage. Therapeutically usefulagents other than the proteins, which may also optionally be included inthe composition, as described above, may alternatively or additionally,be administered simultaneously or sequentially with the composition inthe methods of the invention. In addition, the compositions of thepresent invention may be used in conjunction with presently availabletreatments for cartilage injury such as cartilage allograft orautograft, in order to enhance or accelerate the healing potential ofthe or graft. For example, the, allograft or autograft may be soaked inthe compositions of the present invention prior to implantation. It mayalso be possible to incorporate the protein or composition of theinvention onto suture materials, for example, by freeze-drying.

The compositions may include an appropriate matrix and/or sequesteringagent as a carrier. For instance, the matrix may support the compositionor provide a surface for cartilage-like tissue formation. The matrix mayprovide slow release of the protein and/or the appropriate environmentfor presentation thereof. The sequestering agent may be a substance,which aids in ease of administration through injection or other means,or may slow the migration of protein from the site of application.

The choice of a carrier material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined. Preferred matrices include collagen-based materials,including sponges, such as Helistat.RTM. (Integra LifeSciences,Plainsboro, N.J.), or collagen in an injectable form, as well assequestering agents, which may be biodegradable, for example hyalouronicacid derived. Biodegradable materials, such as cellulose films, orsurgical meshes, may also serve as matrices. Such materials could besutured into an injury site, or wrapped around the tendon/ligament.

Another preferred class of carrier are polymeric matrices, includingpolymers of poly (lactic acid), poly(glycolic acid) and copolymers oflactic acid and glycolic acid. These matrices may be in the form of asponge, or in the form of porous particles, and may also include asequestering agent. Suitable polymer matrices are described, forexample, in W093/00050, the disclosure of which is incorporated hereinby reference.

Preferred families of sequestering agents include blood, fibrin clotand/or cellulosic materials such as alkylcelluloses (includinghydroxyalkylcelluloses), including methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropyl-methylcellulose, and carboxymethylcellulose, the mostpreferred being cationic salts of carboxymethylcellulose (CMC). Otherpreferred sequestering agents include hyaluronic acid, sodium alginate,poly (ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer andpoly(vinyl alcohol). The amount of sequestering agent useful herein is0.5-20 wt %, preferably 1-10 wt % based on total formulation weight,which represents the amount necessary to prevent desorption of theprotein from the polymer matrix and to provide appropriate handling ofthe composition, yet not so much that the progenitor cells are preventedfrom infiltrating the matrix, thereby providing the protein theopportunity to assist the activity of the progenitor cells.

Additional optional components useful in the practice of the subjectapplication include, e.g. cryogenic protectors such as mannitol,sucrose, lactose, glucose, or glycine (to protect the protein fromdegradation during lyophilization), antimicrobial preservatives such asmethyl and propyl parabens and benzyl alcohol; antioxidants such asEDTA, citrate and BHT (butylated hydroxytoluene); and surfactants sifdchas poly(sorbates) and poly(oxyethylenes); etc.

As described above, the compositions and the devices of the inventionmay be employed in methods for enhancing cartilage repair or forinducing cartilage formation. These methods, according to the invention,entail administering to a patient needing such tissue repair; a cellexpresses at least one factor of the T-box family or in anotherembodiment a composition comprising an effective amount of vectorcomprising a nucleic acid encoding a factor of the T-box family.

In another embodiment, as described before, the composition or the cellmay comprise also a vector comprising a nucleic acid encoding FGF and/ora factor of the BMP family.

Preferably the DNA molecule or protein may be injected directly intocartilage tissue such as without limitation nasal cartilage, articularcartilage etc. Therefore, the compounds of the invention may be utilizedas a therapeutic agent in regard to treatment of cartilage or bonedamage caused by disease or aging or by physical stress such as occursthrough injury or repetitive strain, e.g. “tennis elbow” and similarcomplaints. The therapeutic agent of the invention may also be utilizedas part of a suitable drug delivery system to a particular tissue thatmay be targeted.

Other therapeutic applications for the compounds of the invention mayinclude the following: 1. Use in cartilage and/or bone renewal,regeneration or repair so as to ameliorate conditions of cartilageand/or bone breakage, degeneration, depletion or damage such as might becaused by aging, genetic or infectious disease, wear and tear, physicalstress (for example, in athletes or manual laborers), accident or anyother cause, in humans, livestock, domestic animals or any other animalspecies; 2. Stimulation of skeletal development in livestock, domesticanimals or any other animal species in order to achieve increased growthfor commercial or any other purpose; 3. Treatment of neoplasia orhyperplasia of bone or cartilage, in humans, livestock, domestic animalsor any other animal species; 4. Suppression of growth of skeletalcomponents in livestock, domestic animals or any other animal species inorder to achieve decreased growth for commercial or any other purposese.g. by the use of anti sense molecules to the factor of the T-boxfamily; and 5. Alteration of the quality or quantity of cartilage and/orbone for any other purpose in any animal species including humans.

Thus, according to clauses 4 and 5 the invention can be serve also forsuppressing cartilage formation, by the use of an antagonist toBrachyury or to other factors of the T-box family. The antagonisticeffect of dominant negative Brachyury is exemplified in Example 3. Theterm “antagonist” refers to a molecule which, when bound to the epitope,decreases the amount or the duration of the effect of the biological orimmunological activity of epitope. Antagonists may include proteins,nucleic acids, carbohydrates, antibodies, anti sense or any othermolecules, which decrease the effect of Brachyury or to other factors ofthe T-box family on cartilage formation. Such a treatment of suppressionis relevant in the treatment of malignancy of the cartilage, for examplewithout limitation in chondroma and chondrasarcoma. In anotherembodiment the antagonist is a dominant negative factor of the T-boxfamily. In another embodiment the antagonist is a dominant negativeBrachyury.

The term “dominant negative Brachyury refers hereinabove to BrachyuryDNA binding domain (T-box, aa 1-229) without the associated regulatorydomains (aa 230-436).

In another embodiment the invention provides a method of inducingchondrocyte differentiation comprising the step of administering of arecombinant vector, which comprises a nucleic acid encoding a factor ofthe T-box family, thereby inducing chondrocyte formation. Example 2 andFIG. 2 clearly demonstrates that forced expression of the T-box factorBrachyury leads to chondrogenic development in C3H10T1/2 mesenchymalstem cells. Please note that C3H10T1/2 mesenchymal stem cells line.C3H10T1/2 stem cell line resembles human MSCs by many features includingdifferentiation multi potentiality, high proliferation capacity andsimilar response to growth factors and cytokines such as BMPs.

Example 3 further strengthen the relation between Brachyury andchondrogenic development, by demonstrating that dominant negativeBrachyury interferes with BMP2 dependent chondrogenic development inmesenchymal cells.

In another embodiment the invention relates to a method for theproduction of transplantable cartilage matrix, the method comprising thesteps of: obtaining a cell; transfecting said cell with a recombinantvector comprising a nucleic acid sequence encoding a factor of the T-boxfamily, so as to obtain an engineered cell which expresses a factor ofthe T-box family; and culturing said cell with the cell-associatedmatrix for a time effective for allowing formation of a transplantablecartilage matrix. The above method will enable the production of acartilage matrix, which will be transplanted to a subject in need whenrequired.

In another embodiment, there is provided a method of treating a subjectby ex-vivo implantation of at least one cell comprising the followingsteps: obtaining at least one cell from the subject; transfecting thecell with a nucleic acid which encodes at least one factor of the T-boxfamily, so as to obtain an cell which express at least one factor of theT-box family activated cell; and administering said activated cell tothe subject.

Optionally, the enriched stem cells are then expanded ex vivo byculturing them in the presence of agents that stimulate proliferation ofstem cells. The culturing step can be for example in a bioreactor, whichenables three dimensional growth of the cells. The enriched andoptionally expanded stem cells are then infected with a vector, thatexpresses the at least one factor of the T-box family gene. Optionally,the vector may also carry an expressed selectable marker, in which casesuccessfully transduced cells may be selected for the presence of theselectable marker. The transduced and optionally selected stem cells arethen returned to the patient defective connective tissue and allowed toengraft themselves into the bone marrow.

One ex vivo method of enhancing repair and/or inducing formationdisclosed throughout this specification comprises initially generating arecombinant viral or plasmid vector which contains a DNA sequenceencoding a protein or biologically active fragment thereof. Thisrecombinant vector is then used to infect or transfect a population ofin vitro cultured connective tissue cells, resulting in a population ofconnective cells containing the vector. These connective tissue cellsare then transplanted to a target joint space of a mammalian host,effecting subsequent expression of the protein or protein fragmentwithin the joint space. Expression of this DNA sequence of interest isuseful in substantially reducing at least one deleterious jointpathology associated with a connective tissue disorder.

It will be understood by the artisan of ordinary skill, that the sourceof cells for treating a human patient is the patient's own connectivetissue cells, such as autologous fibroblast cells. In another embodimentthe source of cells can be allogenic cells, which were treated so as toreduce immune response.

As used herein, a “promoter” can be any sequence of DNA that is active,and controls transcription in a eucaryotic cell. The promoter may beactive in either or both eucaryotic and procaryotic cells. In anotherembodiment, the promoter is active in mammalian cells. The promoter maybe constitutively expressed or inducible. In another embodiment, thepromoter is inducible. In another embodiment, the promoter is inducibleby an external stimulus. In another embodiment, the promoter isinducible by hormones or metals. Still more in another embodiment, thepromoter is inducible by heavy metals. In another embodiment, thepromoter is a metallothionein gene promoter. In another embodiment thepromoter is inducible by antibiotics such as tetracycline. In anotherembodiment the promoter is inducible by a tissue specific promoter.Likewise, “enhancer elements”, which also control transcription, can beinserted into the DNA vector construct, and used with the construct ofthe present invention to enhance the expression of the gene of interest.

In another embodiment there provided ex vivo and in vivo techniques fordelivery of a DNA sequence of interest to the connective tissue cells ofthe mammalian host. The ex vivo technique involves culture of targetconnective tissue cells, in vitro transfection of the DNA sequence, DNAvector or other delivery vehicle of interest into the connective tissuecells, followed by transplantation of the modified connective tissuecells to the target joint of the mammalian host, so as to effect in vivoexpression of the gene product of interest.

Alternatively, an allograft, (e.g., cartilage grown in vitro fromcartilage tissue removed from the patient) may be implanted by attachinga periosteum membrane (harvested, e.g., from the patient's tibia), tothe bone surface and injecting the allograft beneath the membrane.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer.

Alternatively, the gene encoding the product of interest can beassociated with liposomes and injected directly into the host, such asin the area of the joint, where the liposomes fuse with target cells,resulting in an in vivo gene transfer to the connective tissue. Inanother embodiment, the gene encoding the product of interest isintroduced into the area of the joint as naked DNA. The naked DNA entersthe target cells, resulting in an in vivo gene transfer to the cells.

The dosage of the treatment, which is the amount of the cells whichexpress the at least one factor of the T-box family or in anotherembodiment the amount of the composition or the device which contain thevector comprising the nucleic acid encoding the same in the in vivo andin the ex-vivo treatment the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the composition, e.g., amount of bone or cartilage tissue desired tobe formed, the site of the bone or cartilage damage, the condition ofthe damaged cartilage or bone, the size of a wound, type of damagedtissue, the patient's age, sex, and diet, the severity of anyinfection,. time of administration and other clinical factors. Thedosage may vary with the type of matrix used in the reconstitution andthe types of additional proteins in the composition. The addition ofother known growth factors, such as IGF-I (insulin like growth factorI), to the final composition, may also affect the dosage.

Progress can be monitored by periodic assessment of cartilage formation,and/or repair. The progress can be monitored by methods known in theart, for example, X-rays (CT), ultra-sound, MRI, arthroscopy andhistomorphometric determinations.

In another embodiment, as is exemplified in Example 1 the inventionprovides a method of screening candidate nucleic acid sequence which isinvolved in the early stages of cartilage development, said methodcomprising the step of: obtaining a cell; transfecting said cell with avector comprising a nucleic acid sequence encoding to FGFR3; obtainingmRNA from said cell; synthesizing cDNA from said mRNA; amplifying saidcDNA-hybrid, so as to obtain an amplified product; detecting saidamplified product; and comparing said amplified products from saidsample to amplified products derived from known samples therebyidentifying candidate nucleic acid sequence which is involved in theearly stages of cartilage development.

The term “involved in the early stages of cartilage development” refershereinabove to any gene, which is either upregulated on downregulatedduring the stage of differentiation into a cartilage cell. Such geneswill enable development of drugs which will ether enhance or suppresscartilage formation or repair.

The step of “synthesizing” refer to step of building cDNA complementaryto the mRNA template. As refer hereinabove and in the claims section,the step of “amplifying” refer to the selective replication of a cDNA ingreater number than usual. As refer herein above and in the claimssection, the step of “separating” refer to the step of separation of theproducts using for example, gel electrophoresis. As refer hereinaboveand in the claims section, the step of “detecting” refer to the step ofnoticing, which is done, for example by visualization of the amplifiedproduct's bands. As refer hereinabove and in the claims section, thestep of “comparing” refers to the step of searching for differencesbetween the amplified products derived from the at least two samples.The term “RNA” refers to an oligonucleic in which the sugar is ribose,as opposed to deoxyribose in DNA. RNA is intended to include any nucleicacid, which can be entrapped by ribosomes and translated into protein.The term “mRNA” refers to messenger RNA.

RNA can be extracted from cells or tissues according to methods known inthe art. In a preferred embodiment, RNA can be extracted from monolayersof mammalian cells grown in tissue culture, cells in suspension or frommammalian tissue. RNA can be extracted from such sources by, e.g.,treating the cells with proteinase K in the presence of SDS. In anotherembodiment, RNA is extracted by organic solvents. In yet anotherembodiment, RNA is extracted by differential precipitation to separatehigh molecular weight RNA from other nucleic acids. RNA can also beextracted from a specific cellular compartment, e.g., nucleus or thecytoplasm. In such methods, the nucleus is either isolated forpurification of RNA therefrom, or the nucleus is discarded forpurification of cytoplasmic RNA. Further details regarding these andother RNA extraction protocols are set forth, e.g., in Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989).

EXAMPLES Experimental Procedures

DNA constructs and Transient Transfections

For the assessment of the transcriptional activity a dimmer of thedouble-stranded oligonucleotide of the Brachyury binding element (BBE)AATTFCACACCTAGGTGTGAAATT (Kispert et al., 1995) was incorporated in theBamHI site before the HSV thymidine kinase minimal promoter fused to thecloramphenicol acetyltransferase (CAT)-reporter of pBLCAT5 (Boshart etal., 1992) to give reporter plasmid pBBE-CAT5. 20 h before transfection,human embryonic kidney HEK293T cells were plated at a density of1×104/cm2 in 6-well plates and allowed to grow under normal cultureconditions. For co-transfection experiments, 250 ng per well ofBrachyury expression vector and 250, 500 or 750 ng of the expressionvector encoding dnBrachyury. Empty vector was added to adjust the amountof expression plasmids at 1 ug/ml. 260 ng of BBE-CAT reporter(pBBE-CAT5) was added in the presence of 140 ng of RSV-lacZ vector usingthe DOSPER procedure (see below). Cells were allowed to incubate for 48h. Then, cells were collected and b-galactosidase assays were performedwith the chemiluminescent b-gal reporter gene assay (Roche Diagnostics,Mannheim, Germany) and CAT-assays were carried out with the CAT ELISAkit (Roche Diagnostics, Mannheim, Germany). b-gal assay results wereused to normalize the CAT assay results for transfection efficiency. AllDNA transfection experiments were repeated at least three times intriplicate.

Cell Culture and permanent Transfections

Human embryonic kidney cells HEK293T and murine C3H10T1/2 progenitorcells were routinely cultured in tissue culture flasks in Dulbecco'smodified Eagle's medium supplemented with 10% heat-inactivated FCS, 0.2mM L-glutamine, and antibiotics (50 units/ml penicillin, 50 mg/mlstreptomycin). Cells were transfected using DOSPER according to themanufacturer's protocol (Roche Diagnostics, Mannheim, Germany).C3H10T1/2 cells which recombinantlly express BMP2 (C3H10T1/2-BMP2) cellswere obtained by co-transfection with pSV2pac followed by selection withpuromycin (2.5 ug/ml). FGFR3, Brachyury and T-box domain werePCR-amplified and cloned into expression vectors pMT7T3 and pMT7T3-pgkvectors which are under the control of the LTR of the myeloproliferativevirus or of the murine phosphoglycerate kinase promoter-1, respectively(Ahrens et al., 1993). The integrity of the constructs was confirmed bysequencing. HA-tags were carboxyterminally added to full-lengthBrachyury and Brachyury's T-box domain by PCR with primers encoding therespective peptide sequence. Stable expression of the DNA binding T-boxdomain (aa 1-229) and of the dominant-negative human FGFR3 without thecytoplasmatic tyrosine kinase domains (aa 1-414) in the C3H10T1/2-BMP2background was done by co-transfection with pAG60, conferring resistanceto G418 (750 ug/ml). Individual clones were picked, propagated, andtested for recombinant FGFR3, dnFGFR3, Brachyury or T-box domain(dnBrachyury) expression by RT-PCR (see below). Selected cell cloneswere subcultivated in the presence of puromycine or puromycine/G418 andthe selective pressure was maintained during subsequent manipulations.C3H10T1/2 cells were cultured in DMEM containing 10% fetal bovine serum.The features of C3H10T1/2-BMP2 cells have been described (Ahrens et al.,1993; Hollnagel et al., 1997; Bächner et al., 1998). For the assessmentof in vitro osteo-/chondrogenic development, cells were plated at adensity of 5-7.5×103 cells/cm2. After reaching confluence (arbitrarilytermed day 0) ascorbic acid (50 ug/ml) and 10 mM b-glycerophosphate wereadded as specified by Owen et al., 1990 (1990).

BMP2 inductions

For BMP2-stimulation studies, C3H10T1/2 cells were plated at a densityof 1×104 per cm² in a 9-cm culture dish. After 48 h cells were washed 3×with PBS and then cells were starved for 24 h in DMEM without serum.Before induction the medium was replaced with fresh DMEM without serum.Cells were then treated for the indicated times using recombinant BMP2from E. coli (50 ng/ml). Cycloheximide (50 ug/ml) treatment started 30min prior to the addition of BMP2.

RNA preparation and RT-PCR

Total cellular RNAs were prepared by TriReagentLS according to themanufacturer's protocol (Molecular Research Center Inc.). Five ug oftotal RNA was reverse transcribed and cDNA aliquots were subjected toPCR. RT-PCR was normalized by the transcriptional levels of HPRT. TheHPRT-specific 5′ and 3′ primers were GCTGGTGAAAAGGACCTCT andAAGTAGATGGCCACAGGACT, respectively. The following 5′ and 3′ primers wereused to evaluate osteo/chondrogenic differentiation: SEQ ID. No. 3:collagen 1a1: GCCCTGCCTGCTTCGTG, SEQ ID. No. 4: CGTAAGTTGGAATGGTTTTT;collagen 2a1: SEQ ID. No. 5: CCTGTCTGCTTCTTGTAAAAC, SEQ ID. No. 6:AGCATCTGTAGGGGTCTTCT; SEQ ID. No. 7: osteocalcin: GCAGACCTAGCAGACACCAT,SEQ ID. No. 8: GAGCTGCTGTGACATCCATAC; PTH/PTHrP-receptor: SEQ ID. No. 9:GTTGCCATCATATACTGTTTCTGC, SEQ ID. No. 10: GGCTTCTTGGTCCATCTGTCC; FGFR3:SEQ ID. No. 11: CCTGCGCAGTCCCCCAAAGAAG; SEQ ID. No. 12:CTGCAGGCATCAAAGGAGTAGT; FGFR2: SEQ ID. No. 13: TTGGAGGATGGGCCGGTGTGGTG,SEQ ID. No. 14: GCGCTTCATCTGCCTGGTCTTG. The primer pairs for Brachyuryand Sox9 have been described in (Johansson and Wiles, 1995) and(Zehentner et al., 1999), respectively. Vector-borne transcripts forBrachyury were evaluated with nested primers sets with either vectorspecific 5′- or 3′-primers: SEQ ID. No. 15: TTAGTCTTTTTGTCTTTTATTTCA;SEQ ID. No. 16: GATCGAAGCTCAATTAACCCTCAC.

Western blotting

Recombinant cells from petri dishes (13.6 cm diameter) were harvested atdifferent time points before (day B2), at (day 0) and after (days 2, 4,7) confluence. Lysis was in RIPA buffer (1% (v/v) nonidet P-40, 0.1% SDS(w/v), 0.5% sodium deoxycholate in PBS, containing 100 ug/ml PMSF, 2ug/ml aprotinin, and 1 mM Na3VO4). Lysates were centrifuged (30 min,10.000 g, 4 C) and the supernatants were stored at −70 C until analysis.Protein concentration of the lysates was determined using coomassiebrilliant blue. Protein was precipitated with ethanol, resuspended inreducing (containing DTT) or non-reducing sample buffer and subjected toSDS-gel electrophoresis in 12.5%T polyacrylamide gels (20 ug/lane).Proteins were transferred to nitrocellulose membranes bysemidry-blotting. Protein transfer was checked by staining of themembranes with Ponceau S. After blocking, membranes were incubatedincubated overnight at 4 C with a polyclonal antibody to the HA-tag(SC-805, Santa Cruz Biotechnology, Santa Cruz, Calif.) diluted 1:200 inblocking solution. FGFR3 and FGFR2 antibodies were from Santa CruzBiotechnology (#SC-123, #SC-122; Santa Cruz, Calif.). The secondaryantibody (Dianova, Hamburg) was applied at 1:5000 in blocking solutionfor 2 h at room temperature. Color development was performed with4-chloro-1-naphthol and H2O2.

Histological Methods and Verification of Cellular Phenotypes

Osteoblasts exhibit stellate morphology displaying high levels ofalkaline phosphatase, which was visualized by cellular staining withSIGMA FAST BCIP/NBT (Sigma, St. Louis, Mo.). Proteoglycan secretingchondrocytes were identified by staining with Alcian Blue at pH 2.5 andstaining with Safranin O (Sigma, St. Louis, Mo.). Forcollagen-immunohistochemistry cells were washed with PBS and fixed withmethanol for 15 min at −20 C by methanol. Primary antibodies werediluted with 1% goat serum in PBS. Monoclonal anti-collagen IIantibodies (Quartett Immunodiagnostika, Berlin, Germany, # 031502101)were diluted 1:50 and monoclonal anti-collagen X antibodies (QuartettImmunodiagnostika, Berlin, Germany, # 031501005) 1:10, respectively.Incubation was for 1 hour at room temperature followed by staining withZymed HistoStain SP kit (Zymed Laboratories Inc., San Francisco, Calif.)applying the manufacturer's protocol. A positive signal is indicated bya red color precipitate of AEC (aminnoethylcarbazole).

In Vivo Transplantation

Before in vivo transplantation, aliquots of 2-3×106 cells were mountedon individual type I collagen sponges (Colastat7 #CP-3n, VitaphoreCorp., 2×2×4 mm.) and transplanted into the abdominal muscle of femalenude mice (4-8 weeks old). Before transplantation animals wereanaesthetized with ketamine-xylazine mixture 30 ul/per mouse i.p. andinjected i.p. with 5 mg/mouse of Cefamzolin (Cefamezin7, TEVA). Skin wasswabbed with chlorhexidine gluconate 0.5%, cut in the middle abdominalarea, an intramuscular pocket was formed in a rectal abdominal muscleand filled with the collagen sponge containing cells. Skin was suturedwith surgical clips. For the detection of engrafted C3H10T1/2 cells themice were sacrificed 10 days and at 20 days after transplantation.Operated transplants were fixed in 4% paraformaldehyde cryoprotectedwith 5% sucrose overnight, embedded, and frozen. Sections were preparedwith a cryostat (Bright, model OTF) and stained with H&E, Alcian Blueand Safranin O.

RNA-In Situ-Hybridization

Embryos were isolated from pregnant NMRI mice at day 18.5 postconceptionem (dpc). The embryos were fixed overnight with 4%paraformaldehyde in PBS at 4 C. 10 um cryosections were mounted onaminopropyltriethoxysilane coated slides and non-radioactive RNA-insitu-hybridizations were done as described (Bächner et al., 1998) and byfollowing the instructions of the manufacturer (Roche, Mannheim). Inshort: For hybridization sense- and antisense RNA probes from a 1.8 kbmurine Brachyury cDNA was used. For the generation of collagen 1a1 orcollagen 2a1 the vector pMT7T3 was used harbouring specific probes(Metsäranta et al., 1991). Hybridization was performed with 0.5-2 ugdenatured riboprobe/ml) over night at 65 C in a humid chamber. Fordigoxygenin (DIG)-detection slides were blocked in 5× SSC, 0.1% Triton,20% FCS for 30 minutes following two washes with DIG-buffer 1 (100 mMTris, 150 mM NaCl, pH 7.6) for 10 minutes. Slides were incubated inanti-DIG-alkaline phosphatase coupled antibodies diluted 1:500 inDIG-buffer 1 over night in a humid chamber. Slides were washes with 0.1%Triton in DIG-buffer 1 for 2 hours with several changes of the washingsolution and equilibrated in DIG-buffer 2 (100 mM Tris, 100 mM NaCl, 50mM MgCl2). Detection was performed using BM-purple substrate (Roche,Mannheim) in DIG-buffer 2 with 1 mM Levamisole for 1-6 hours dependingon the probe. Reaction was stopped in TE-buffer and slides wereincubated in 3% paraformaldehyde in PBS for 3 minutes, in 0.1 M glycinein PBS for 3 minutes and washed three times in PBS for 3 minutes. Slideswere counterstained with 0.5% methylenegreen in PBS for 1 minute,dehydrated in graded alcohol series, air-dried and mounted with Eukitt.

EXPERIMENTAL RESULTS Example 1

BMP2-dependent Chondrogenic Development in C3H10T1/2 MSCs InvolvesFGF-Receptor 3

During a substractive screen for BMP-regulated genes in recombinantBMP2-expressing C3H10T1/2 (C3H10T1/2-BMP2) cells upregulation of theFibroblast Growth Factor Receptors 3 and 2 (FGFR3, FGFR2) was noted atboth the transcriptional and protein levels (FIG. 1 a, c, respectively).These two receptor types exhibit different induction kinetics. FGFR3 isupregulated during early stages of cultivation in the stableC3H10T1/2-BMP2 line while FGFR2 shows a delayed response (FIG. 1 a, c).The fast upregulation of FGFR3 seems to be due to an immediate responseto BMP2 since exogenously-added BMP2 mediated FGFR3 transcription inwild-type C3H10T1/2 cells in the presence of cycloheximide (FIG. 1 b).In contrast to FGFR3 and FGFR2 is FGFR1 constitutivelly expressed inwild type and C3H10T1/2-BMP2 cells (FIG. 1 a) while FGFR4 does not showany significant rates of expression. Since FGFs and their receptors arecrucial modulators of chondrogenic development, an assessment of whetherthe immediate BMP2-dependent upregulation of FGFR3 in C3H10T1/2 isinvolved in the onset of chondrogenic differentiation was conducted.Indeed the results demonstrated that forced expression of the wild-typeFGFR3 (FGFR3WT) was sufficient for the development of morphologicallydistinct chondrocytes in C3H10T1/2-FGFR3WT cells (FIG. 1 d, e).Moreover, the constitutively active mutant FGFR3 (Ach, G380R) possessesthe same capacity. The forced expression of FGFR3WT in MSCs stimulatesMAPK signaling in these cells as documented by enhanced levels of ERK1and ERK2 phosphorylation (right panels in FIG. 1 d), leads to thedevelopment of histologically distinct chondrocytes and induces orincreases expression of chondrogenic marker genes such as collagen 2a1,the PTH/PTHrP receptor and transcription factor Sox9 (FIG. 1 e).

The immediate BMP2-dependent upregulation of FGFR3 in MSCs (C3H10T1/2)and the inherent capacity of this receptor to initiate chondrogenicdevelopment in these cells prompted a screen for FGFR3-regulatedtranscription factors. It was observed that among the transcriptionfactors tested, the T-box transcription factor Brachyury was upregulatedin FGFR3-expressing C3H10T1/2 cells (see also FIG. 5 a). Thereupon, thechondrogenic potential that Brachyury possesses after recombinantexpression in wild-type C3H10T1/2 cells (see below) was demonstrated.

Example 2

Forced Expression of the T-box Factor Brachyury Leads to ChondrogenicDevelopment in C3H10T1/2 Mesenchymal Stem Cells

Brachyury has originally been described as the first member of a familyof transcription factors that harbors a T-box as the DNA-binding domain.In order to assess that the FGFR3-dependent upregulation of the T-boxfactor Brachyury in C3H10T1/2 plays a role in chondrogenesis BrachyurycDNA was expressed under the control of the murine phosphoglyceratekinase-1 (PGK-1) in mesenchymal stem cell line C3H10T1/2 to allowmoderate expression levels of Brachyury (C3H10T1/2-Brachyury). Therecombinant expression of Brachyury cDNA under the control of the murinephosphoglycerate kinase-1 (PGK-1) in MSCs (FIG. 2 a) gave rise toefficient chondrogenic differentiation resulting in alkaline phosphatasepositive cells (beginning at day 4) and Alcian Blue positivechondrocyte-like cells (at day 10 post-confluence; FIG. 2 b). Threeindividual C3H10T1/2 clones were investigated in regard to theirchondrogenic potential and gave similar results. Immunohistochemistryconfirms the presence of the chondrocyte-specific collagen 2 but not ofcollagen X which is typical for late stages of chondrocyticdifferentiation (hypertophic chondrocytes) (FIG. 2 b, left panel). Majormarker genes of chondrogenic and, also, of osteogenic development show atransient (collagen 2a1, PTH/PTHrP-receptor) or permanent upregulation(osteocalcin gene and the chondrogenic transcription factor Sox9) inC3H10T1/2-Brachyury in comparison with C3H10T1/2 cells which were stablytransfected with an empty expression vector (FIG. 2 c). Although, theinduction of the osteocalcin gene indicates an osteogenic potential forC3H10T1/2-Brachyury, ectopic transplantation of these cells in murineintramuscular sites results exclusively in the massive formation ofproliferating chondrocytes and cartilage (FIG. 2 d). These ectopictransplantations have been performed three times and in all cases thesetransplants developed chondrocytes and cartilage. After both 10 and 20days transplants exhibit a histological presence of proteoglycans(Alcian Blue, Safranin O) while bony elements or mineralized particlesare not observed (FIG. 2 d). After 20 days the ectopic implants showareas of extensive extracellular matrix production as visualized byhistological analyses (FIG. 2 d). The use of stronger viral promoterssuch as the LTR of the myeloproliferative virus (MATERIALS and METHODS)resulted in increased cellular proliferation without the apparentformation of histologically distinct mesenchymal cell types.

Example 3

Dominant-negative Brachyury Interferes with BMP2-dependent ChondrogenicDevelopment in MSCs.

A partial nuclear localization signal (NLS) which has been attributed tothe T-box domain should allow a substantial nuclear accumulation(Kispert et al., 1995). The dominant-negative nature of the T-box domainwas confirmed in DNA co-transfection assays performed in HEK293 T cells.This cell line does not express Brachyury. Exogenous Brachyurytransactivated a construct containing two copies of the consensusBrachyury binding element (BBE) oligonucleotide fused to a minimal HSVthymidine kinase (TK)-minimal promoter-CAT chimeric gene, pBBE(2×)-CAT5(FIG. 3 a). Indeed, co-transfection of pBBE(2×)-CAT5 with a recombinantBrachyury-expressing vector resulted in a 25-fold activation, whereas anempty expression vector had no effect (FIG. 3 a). Co-transfection offull-length Brachyury (Brachyury wt) with increasing amounts of anexpression vector expressing the T-box domain (dnBrachyury) (1:1, 1:2,1:3) led to a clear decrease in CAT (7-fold). Exogenous dnBrachyuryalone transactivated pBBE(2×)-CAT5 (BBE) only 3-fold.

The forced expression of the HA-tagged T-box domain (dnBrachyury) isobserved throughout in vitro cultivation (FIG. 3 b) and stronglyinterfered with the BMP2-mediated formation of alkaline phosphatasepositive osteoblast-like and Alcian Blue chondrocyte-like cells in vitro(FIG. 3 e). In vivo, in ectopic transplantations of C3H10T1/2-BMP2 inintramuscular sites, dnBrachyury allowed the development of connectivetissue only (FIG. 3 e). In addition, the chondrocyte-specific collagen2a1 mRNA levels are more sensitive to the presence of dnBrachyury thanmRNA levels of the distinct osteogenic marker osteocalcin. The latter ishardly affected, consistent with the idea that Brachyury possesses apredominant chondrogenic capacity in this particular cell type.Interestingly, the BMP2-mediated transcriptional upregulation of FGFR3in C3H10T1/2 is not obstructed by dnBrachyury indicating that theimmediate BMP2-mediated FGFR3 induction is independent of Brachyury orother T-Box factors (FIG. 3 c, d). However, FGFR2-expression that bits adelayed response in C3H10T1/2-BMP2 cells (FIGS. 1 and 3) displays a highsensitivity to dnBrachyury. BMP-mediated FGFR2 expression is almostcompletely suppressed by the dominant-negative acting T-box domain (FIG.3 c, d). This may indicate that that the presence of FGFR2 seemsnecessary for the osteo-/chondrogenic differentiation in thismesenchymal progenitor line (FIG. 3 c, d).

Furthermore, this suggests a hierarchy of FGFR-mediated signaling forchondrogenic development. FGFR3-dependent signaling is induced at firstby BMP2 and as a consequence, FGFR2 mediated signaling becomes active.Such a model is proposed in FIG. 7. This model predicts that a forcedexpression of dominant-negative FGFR3 would interfere with BMP2-mediatedchondrogenesis and with FGFR2 and Brachyury expression. Indeed, anFGFR3-variant without the cytoplasmatic tyrosine-kinase domainsdownregulates BMP2-dependent mRNA expression levels of FGFR2 andBrachyury (FIG. 4 b) and interferes with the histological manifestationof alkaline phosphatase or Alcian Blue positive chondrocyte-like cells(FIG. 4 a).

Example 4

FGFR3 and the T-box Factor Brachyury Are Involved in an AutoregulatoryLoop for Chondrogenic Development in C3H10T1/2 Progenitors

During amphibian gastrulation, mesodermal Brachyury is involved in anautoregulatory loop with FGF that is present in the embryo (1998). InC3H10T1/2 cells several FGF genes tested (FGF2, 4, and 9) were notBrachyury- or FGFR3-regulated and, therefore, are unlikely members ofsuch a loop. However, a loop seems to exist between FGFR3 and Brachyurysince forced expression of either one lead to the induction of the otherone in C3H10T1/2 (FIG. 5 a). These experiments indicate that afterBMP2-mediated initiation of the chondrogenic lineage, the chondrogenicdifferentiation may advance for some time in a BMP2-independent fashionmaintained by the autoregulatory loop between FGFRs and FGF-regulatedtranscription factors such as the T-box factor Brachyury.

In a preceding study it was shown that BMP-mediated R-Smad signalingalone is not sufficient for cartilage development in C3H10T1/2 cells.Thereby, forced expression of Smad1 or the biologically active Smad1-MH2domain is able to mimic BMP2-mediated onset of osteogenicdifferentiation (Takeuchi et al., 2000). However, in contrast toosteogenic marker genes such as the osteocalcin gene, Smad1-MH2domain-signaling is not sufficient to mimic BMP2-dependent FGFR3- andthe concomitant Brachyury-gene induction (FIG. 5 b). Other BMP-activatedR-Smads such as Smad5 and Smad8 are also unable to mediate or to mimicBMP2-dependent FGFR3-induction in C3H10T1/2 cells indicatingR-Smad-MH2-independent pathways for FGFR3 induction or, alternatively,cooperative activities of R-Smads with other transcription factors(Mazars et al., 2000).

Example 5

The T-box Factor Brachyury Is Expressed in Maturing Cartilage DuringMurine Embryonic Development

Brachyury is expressed at high levels early in vertebrate embryonicdevelopment and is involved in gastrulation and in the dose-dependentdetermination of mesodermal cell fates. After gastrulation,Brachyury-expression is downregulated and persists in the notochord tothe end of embryogenesis (Kispert and Herrmann, 1994). Comparative mRNAexpression analysis of murine Brachyury (Bra), collagen 1a1 (col 1a1)and collagen 2a1 (col 2a1) in skeletal development (18.5 dpc) indicatesthat Brachyury is expressed at significant levels in cartilage formingcells of the intervertebral disks and in limb bud development (FIG. 6).Expression of Brachyury is enhanced in intervertebral disc developmentin the nucleus pulposus in 18.5 dpc mouse embryos (FIG. 6A, a, d)confirming earlier reports (Wilkinson et al., 1990). Collagen 1a1 isexpressed in the outer annulus (arrowheads in FIG. 6A b, e), andcollagen 2a1 in the cartilage primordium of the vertebrae (FIG. 6A, c,f). In transversal sections made at the level of the upper lumbarvertebra, expression of Brachyury is in addition detectable in distinctchondrogenic cells of the neural arch (FIG. 6A, h) whereas collagen 1a1expression is maintained in the outer annulus (FIG. 6A, i), as iscollagen 2a1 in the cartilage primordium (FIG. 6A, j). In murine limbbud development (18.5 dpc; hind limb) expression of Brachyury is evidentin distinct chondrogenic cells of the forming metatarsal bones (FIG. 6B,a-c). In contrast, collagen 1a1 is expressed in the outer periosteallayer (FIG. 6B, d-f) and collagen 2a1 expression is enhanced indifferentiating chondrocytes (FIG. 6B, g-i). Interestingly, like inintervertebral disc formation, the expression of Brachyury is onlyevident in chondrocyte-like cells that do not express Col 2a1 indicatingthat Brachyury expression is upregulated in chondrogenic cells before orafter collagen 2 expressions.

Example 6

The T-box Factor Brachyury Is Expressed in Human Adult Mesenchymal Cellsfollowing transfection with Brachyury plasmid.

Cells isolation:

Human Adult Mesenchymal Stem Cells (hAMSCs) were isolated from explantsof human bone marrow surgical waste and expanded in vitro. Isolation ofhMSCs was performed as follows: 10 ml marrow aspirates were collectedinto a tube with 6000 U heparin, washed with PBS, and recovered cellswere collected by centrifugation at 900 g. Collected cells were thenloaded onto Percoll solution (density 1.073 g/ml). Cell separation wasaccomplished by centrifugation at 1100 g (30 min at 20 uC). Nucleatedcells collected were washed twice with PBS and then cultured in 100 mmculture plates.

Tissue culture:

Cells were cultured in low glucose, low bicarbonate DMEM medium (BeitHaemek)+10% fetal calf serum (Biet Haemek), the environmental conditionswere of 5% CO2 and 370 C.

Cells transfection:

3×10⁶ hAMSCs were transfected with 30 ug of the Brachyury plasmid usingthe Amaxa Nucleofector™ technology and in accordance with themanufacturer's preliminary protocol for hAMSCs. Briefly, the harvestedcells were aliquoted in 5×10⁵ cells, recovered by centrifugation, andre-suspended in 100 μl of Amaxa's nucleofection solution. Five microgramof DNA plasmid were added to the suspended cells, mixed well andtransferred to electroporation cuvette provided by the Amaxanucleofection kit. The electroporation was performed using 5 differentprograms (U28, C12, C17, E14, G22) that basically differ in theintensity and the length of the electric pulse. Immediately after theelectroporation, the cells were transferred into 6-well plates,containing 4 ml complete growth medium equilibrated to 37 C, 5% CO2, andincubated at 37° C. in 5% CO₂ atmosphere for 24 hours.

Detection of gene expression:

24 hours post transfection RNA was isolated from the cells using theTrizol reagent and protocol provided by the manufacturer (LifeSciences). 2 ug of RNA were transformed into cDNA by ReverseTranscriptase (RT) reaction. PCR was then performed using specificprimers to the Brachyury cDNA. 20 ul of the PCR reaction sample wereloaded into a 2% Agarose gel stained with Etidium Bromide. The gelanalysis demonstrated a band matching the expected amplified region inthe Brachyury cDNA (see FIG. 8).

1-10. (canceled)
 11. A method of enhancing repair of a cartilage in thebody comprising the step of administrating a recombinant vector whichcomprises a nucleic acid encoding a factor of the T-box family to thecartilage of a subject, thereby enhancing repair of the cartilage. 12.The method of claim 11, wherein said factor of the T-box is brachyury.13. The method of claim 11, wherein said method further comprisesadministering a recombinant vector which comprises a nucleic acidencoding a factor which upregulates the expression of the T-boxtranscription factor.
 14. The method of claim 13, wherein said factorwhich upregulates the expression of the factor of the T-box is FGF orBMP2.
 15. A method of inducing formation of a cartilage in the bodycomprising the step of administrating a recombinant vector whichcomprises a nucleic acid encoding a factor of the T-box family to thecartilage of a subject, thereby inducing formation of the cartilage. 16.The method of claim 15, wherein said factor of the T-box is brachyury.17. The method of claim 15, wherein said method further comprisesadministering a recombinant vector which comprises a nucleic acidencoding a factor which upregulates the expression of the T-boxtranscription factor.
 18. The method of claim 17, wherein said factorwhich upregulates the expression of the factor of the T-box is FGF orBMP2.
 19. A method of inducing chondrocyte differentiation comprisingthe step of administering of a recombinant vector which comprises anucleic acid encoding a factor of the T-box family, thereby inducingchondrocyte formation.
 20. The method of claim 19, wherein said factorof the T-box is brachyury.
 21. The method of claim 19, wherein saidmethod further comprises administering a recombinant vector whichcomprises a nucleic acid encoding a factor which upregulates theexpression of the T-box transcription factor.
 22. The method of claim19, wherein said factor which upregulates the expression of the factorof the T-box is FGF or BMP2. 23-48. (canceled)
 49. A compositioncomprising at least one recombinant vector which comprises a nucleicacid sequence encoding at least one factor of the T-box family and apharmaceutically acceptable carrier.
 50. The composition of claim 49,wherein said composition is a pharmaceutically composition.
 51. Thecomposition of claim 49, wherein said factor of the T-box is brachyury.52. The composition of claim 49, wherein said method further comprisesadministering a recombinant vector which comprises a nucleic acidencoding a factor which upregulates the expression of the T-boxtranscription factor.
 53. The composition of claim 52, wherein saidfactor which upregulates the expression of the factor of the T-box isFGF or BMP2. 54-66. (canceled)